key: cord-0804048-lyag1mx7 authors: Mendonça, Alexandre; Santos, Helena; Franco-Duarte, Ricardo; Sampaio, Paula title: Fungal Infections Diagnosis – Past, Present and Future date: 2021-12-01 journal: Res Microbiol DOI: 10.1016/j.resmic.2021.103915 sha: e9e1a2d5799cba1b52b94fab77d670256fab5a45 doc_id: 804048 cord_uid: lyag1mx7 Despite the scientific advances observed in the recent decades and the emergence of new methodologies, the diagnosis of systemic fungal infections persists as a problematic issue. Fungal cultivation, the standard method that allows a proven diagnosis, has numerous disadvantages, as low sensitivity (only 50% of the patients present positive fungal cultures), and long growth time. These are factors that delay the patient’s treatment and, consequently, lead to higher hospital costs. To improve the accuracy and quickness of fungal infections diagnosis, several new methodologies attempt to be implemented in clinical microbiology laboratories. Most of these innovative methods are independent of pathogen isolation, which means that the diagnosis goes from being considered proven to probable. In spite of the advantage of being culture-independent, the majority of the methods lack standardization. PCR-based methods are becoming more and more commonly used, which has earned them an important place in hospital laboratories. This can be perceived now, as PCR-based methodologies have proved to be an essential tool fighting against the COVID-19 pandemic. This review aims to go through the main steps of the diagnosis for systemic fungal infection, from diagnostic classifications, through methodologies considered as “gold standard”, to the molecular methods currently used, and finally mentioning some of the more futuristic approaches. are available. Zhang and co-workers [32] analysed the clinical characteristics of 145 cases of 166 invasive fungal infections. Webb and colleagues [33] , analysed the incidence, clinical features and 167 outcomes of invasive fungal infections in the US health care network, according to 3374 episodes 168 in 3154 patients. 169 170 171 The diagnosis of a fungal infection is a lengthy process, especially due to the symptomatic 172 similarities between a bacterial and fungal infection. Figure 1 provides a simplified framework 173 reviewing the steps for the diagnosis of systemic fungal infections. When a patient that presents 174 clinical signs of infections does not respond to an antibiotic treatment ( Figure 1A ), an invasive 175 fungal infection should be included in the patient's differential diagnosis ( Figure 1B-C) . The host 176 factors and clinical signs defined by the EORTC and MSG-ERC are analysed ( Figure 1D ) and 177 methodologies available for mycological evidences should be selected accordingly. There are 178 several methodologies available for mycological evidences but it is important to notice that the 179 type of methodology used will dictate the diagnosis classification ( Figure 1E ). Methodologies that 180 are capable of detecting the fungal pathogen through histopathological or culture methods from 181 sterile sites provide a proven diagnosis. However, if positive mycological evidence is achieved 182 through molecular methodologies, such as PCR-based methods or serological tests, a probable 183 diagnosis is attributed. Still, it is of utmost importance to achieve specific identifications to 184 establish the most appropriate therapeutic plan, which can lead to higher survival rates and decrease 185 hospital costs ( Figure 1F ). 187 For gathering mycological evidences to obtain a definitive proven diagnosis, the fungal 188 culture, microscopy, and histopathology remain the gold standard methods for identification of the 189 J o u r n a l P r e -p r o o f [38] . Additionally, that the vast majority of the blood culture vials took about 14 to 72 hours to 214 become positive [39] . A comparison between C. albicans, C. dubliniensis, C. krusei, C. lusitaniae, 215 C. parapsilosis and C. tropicalis growth times, showed no significant differences regardless of the 216 culture used (40 hours to 57 hours). However, C. glabrata had an overall lower growth time (12 217 hours to 37 hours) and the culture vial was an important factor since significant differences in 218 growth time were observed (Bactec Mycosis IC/F 12 hours, and BacT/Alert FN 37 hours) [38] . 219 Candida spp. in the bloodstream (candidemia) is associated with around 40 to 68% of cases 220 of candiduria (presence of Candida species in the urinary tract) [40, 41] . Therefore, in suspected 221 candidemia, the use of urine cultures can also be considered [42] . Once more the culture medium 222 has a significant effect on the performance of detection. The standard urine culture (blood and 223 MacConkey agars) showed only 37% of detection while the fungal culture medium (Sabouraud 224 dextrose agar) showed detected 98% of Candida spp. in urine [43] . 225 Cerebrospinal fluid samples (CSF) cultivation is often used in suspected fungal infections 226 on the central nervous system but it detects mainly Cryptococcus and Candida [44, 45] . The most 227 appropriate media for pathogens growth from CSF are Sabouraud 4% dextrose and sheep blood 228 agar plates [44] . These pathogens usually take about 3 to 7 days to grow, with very good sensitivity 229 for Cryptococcus, >95%, while for Candida only 37% [35, 46] . However, due to the risk associated 230 with CSF collection, that includes nerve damage, the possibility of an infection, discomfort and/or 231 pain, and bleeding into the spinal cord, CSF culture is not the first option in sample collection [47] . 232 Despite advances in fungal cultures, the majority of moulds are rarely isolated from CSF 233 or blood cultures, however in cases of disseminated infection, the Fusarium spp. blood cultures are 234 often positive [36, 45, 48] . Therefore, particularly for invasive aspergillosis (IA), detection of susceptibility to contamination and low sensitivities values. However, this method provides a 262 proven diagnosis, and it can lead to antifungal susceptible tests and identification at the species 263 level (Table 1) . Direct microscopy is used to look for fungi morphological structures in a portion of infected 267 biopsy tissue or fluid. This allows to evaluate whether the infection is triggered by a septate mould 268 (such as Aspergillus spp.), a non-septate mould (for example Mucorales), or a yeast (for example 269 a Candida spp.) [52] . Throughout the visualization of the fungi's appearance in the tissue section 270 and identification of specific morphological patterns, it is possible to differentiate between different 271 histopathological diagnoses associated with invasive fungal infections. However, the visualization 272 of those structures alone does not provide a specific identification since the analysed structures are 273 similar in various fungal species [53] . Additionally, it is very important to assess tissue invasion to 274 understand the significance of the isolate (pathogenic fungus / normal microbiota / environmental 275 contamination). Visualization of fungal structures by histopathology and direct microscopy 276 techniques can be improved, through the use of stains, such as Gomori's methenamine silver, the 277 periodic acid-Schiff reaction [53] , or India ink [54] , and fluorescent brighteners, such as Calcofluor 278 White [55] . 279 Biological samples from patients with clinical diagnosis of cryptococcosis (CSF samples) 280 can be examined directly for the presence of encapsulated budding yeast by India ink preparation. 281 However, the agreement between direct detection of encapsulated yeast cells with India ink and 282 fungal culture ("gold standard") varies, Martins et al. [56] showed an 80% (8 with India ink test 283 out of 10 positive fungal culture), Sato and colleagues [57] showed only 44% (7 out of 16), while 284 Silva et al. [54] presented 95% ( [53, 58] . Nevertheless, these techniques are very useful to avoid false negative results 296 from the fungal culture or cases of uncultivable fungi and, implementation of a standardized 297 reporting format should improve diagnostic accuracy and prevent adverse outcomes [58] . Still, 298 microscopy requires a trained mycologist to differentiate different species, or even genus, 299 especially due to the similar microscopic appearance of several fungus (Table 1) . The isolation of the fungal pathogen from the cultures allows the evaluation of several 303 relevant parameters as their antifungals resistance and species identification [55] . Despite the 304 growth of the pathogenic fungi, the culture media provides only information about the 305 presence/absence of fungus. Therefore, in order to be able to identify the fungal species behind the 306 infection, there are complementary methodologies to achieve a specific identification, leading to a 307 better therapeutic plan. Considering the unspecific clinical scenarios of fungal infections, the detection of the 311 presence or absence of a fungal pathogen is frequently insufficient, thus chromogenic media can 312 be used to overcome this limitation [59] . Chromogenic media has been widely used in clinical 313 microbiology to detect and identify either bacterial or fungal pathogens [60] being used for 314 Candida identification since 1994. They allow the growth of a specific microorganism, and with 315 the inclusion of chromogenic or fluorescent enzyme subtracts targeting microbial enzymes, such 316 media are able to target pathogens with high specificity [59] . These culture media are suitable for 317 non-sterile samples as they stimulate the growth of a specific genus, inhibiting the growth of other 318 microorganisms [34] . There are several chromogenic media available for the detection of yeasts, 319 and these media often include a chromogenic substrate for β-hexosaminidase, which allows the 320 differentiation of the most frequent and clinically important species, C. albicans. Combining namely C. albicans, C. tropicalis, C. glabrata, C. parapsilosis and C. krusei, on the basis of 328 colouration and colony morphology [59, 61] . Even though chromogenic media can provide rapid and direct identification by colony colour and 330 morphology observation, they were not able to differentiate emerging Candida species, such as C. 331 auris, until very recently. In 2020, Borman et al. [62] conducted a study that aimed to test C. auris 332 detection and specificity of a novel CHROMagar TM Candida Plus, using 52 yeast species 333 J o u r n a l P r e -p r o o f recovered from clinical samples. The authors reported that CHROMagar TM Candida Plus was able 334 to successfully distinguish C. auris from all other species tested due to the distinct appearance of 335 its pale cream colony with a blue halo , except for Candida diddensiae, which showed a similar 336 appearance to C. auris [62] . De and compared with a database. However, before performing these methods, it is necessary to obtain 359 a pure culture of the pathogen [64] . Considering fungal infections, these systems are most suitable 360 for yeast species as for instance the manual API 20C AUX and the automated VITEK ® 2 YST ID 361 card (bioMérieux, Marcy-l'Étoile, France). API 20C AUX system properly identifies around 97% 362 of the most commonly detected species [65, 66] . The accuracy of these two systems (VITEK® 2 and API 20C AUX) for yeast detection is better 364 for commonly detected species (76 -95% for VITEK® and 96 % for API) than for uncommon 365 yeast species (58-78% for VITEK® 2 and 72% for API) [66] [67] [68] [69] . Misidentifications or 366 identifications were mostly detected for C. parapsilosis, C. glabrata, C. dubliniensis, C. 367 norvegensis and C. pelliculosa [66, 67] . VITEK® 2 system database has been recently updated 368 (version 8.01) to include C. auris, increasing the accuracy of identification of this emergent 369 pathogen. However, the updated version showed limited ability to distinguish between C. auris and 370 closely related species, only identifying correctly about 52% of the C. auris isolates [70, 71] . These 371 biochemical assays provide inaccurate identification of C. auris, mistaken this species with for 372 instance C. haemulonii, Rhodotorola glutinis, C. famata, or C. sake [72, 73] . with universal profile databases, enabling identification at the species and genus level [34, 35] . 382 Becker and colleagues [74] [78] . Overall, common yeast and moulds are mostly well covered in commercially 408 available databases, but the same does not happen for new, rarer, or cryptic species. It seems that 409 the best approach to improve the identification of these species is to continuously update the platform [79] . In addition to species-level identification, assessing the antifungal resistance is essential to 414 select the most suitable antifungal therapeutic approach. Several studies have demonstrated that 415 MALDI-TOF MS has the potential to be adapted for antimicrobial susceptibility testing. 416 Vatanshenassan and colleagues recently introduced the MALDI Biotyper antibiotic susceptibility 417 test-rapid assay (MBT ASTRA) to detect echinocandin resistant C. albicans, C. glabrata and C. 418 auris isolates [80, 81] . MALDI-TOF MS is associated with several advantages such as being capable of 420 identifying the pathogen at the genus and species level with accurate and fast results, with easy 421 handling and reduced costs. It is applicable to a vast variety of microorganisms and allows 422 antifungal susceptibility detection. Still, it is incapable of performing quantification, and the 423 coverage of databases available is limited, so there must be a continuous update to cover the rarest 424 and emergent species (Table 1) . krusei and C. glabrata) [83] . A study using this new platform showed that of 137 patients with 448 positive blood cultures without antifungal treatment, the YTL PNA FISH was able to correctly 449 target the treatment of 132 patients (96.4%), and distinguish between bacteria and yeasts in a 450 concomitant growth (95.8%) [83] . These platforms are capable of displaying results within two 451 hours, with high sensitivity and specificity values (above 95%) [82] . In microbiology laboratories this assay was replaced by more efficient methodologies especially because FISH platforms 453 presented a high limit of detection, and there was a reduced number of PNA probes commercially 454 available (Table 1) . specificity regarding Candida species: 93 and 100%, respectively [88] . It also showed excellent 481 results when identifying cultures with more than one pathogen, with a sensitivity of 89%. This 482 diagnostic assay was optimized to use samples directly from the blood culture without previous 483 DNA extraction [88] . Fusarium spp, and Rhodotorula spp. [90, 91] . The sensitivity of ePlex ® system from blood cultures 493 ranged from 99.8 to 100% for fungal pathogens, and specificity of 100% [91, 92] . happen if the PCR-platform does not occur in a closed system, and also the design of primers 498 should be carefully made (Table 1 ). When no detection of the pathogenic fungi through histopathological or culture methods 501 from sterile sites is possible, but only detection of traces of the pathogen, a probable diagnosis is 502 attributed. Serological, molecular, and other more recent techniques are available to collect 503 evidence of the presence of the pathogenic fungi. However, for a probable diagnosis to be 504 attributed, several aspects must be considered, such as the patients' clinical signs and symptoms 505 and the host factors (immunocompromised or not). Regarding mycological evidences, there should 506 be a final result that is concordant between at least two different methodologies. Some of these 507 methodologies can also be used after a positive blood culture for species identification. by the pathogen. MR is the preferred modality, which makes evident the thick meningeal 525 enhancement, at the skull base [93] . Sinus infections are most commonly associated with 526 Mucorales fungi, especially Rhizopus and Rhizomucor species, and Aspergillus spp., which may 527 cause bone destruction, and spread to other areas, as soft tissues and even to intracranial cavity. 528 Initially, CT is often used to analyse bone destruction whereas MR is applied to evaluate the 529 extension of intracranial cavity sinus infections [93] . Concerning pulmonary aspergillosis, the 530 commonly associated organisms are Aspergillus, Candida¸ Nocardia and Actinomyces species, as 531 well as Mucorales fungi, Pneumocystis jirovecii, Histoplasma species and Cryptococcus species. As for pulmonary infections, a variety of characteristics can be observed, such as nodules, chest 533 wall invasion and lobar consolidations. CT is often applied to visualise parenchymal nodules or 534 consolidation with a surrounding area, called CT "halo", linked to angioinvasive pulmonary 535 aspergillosis, Candida spp., Coccidioides and Cryptococcus infections. In contrast, pulmonary 536 mucormycosis is associated to a CT "reverse-halo" (consolidation surrounding a central opacity) 537 [93] . Regarding gastrointestinal and genitourinary infections, the most associated fungal pathogen 538 is C. albicans. When using CT and MR imaging these infections usually manifest similar 539 parenchymal microabscesses [93] . 540 The imaging methodologies commonly used to assess and diagnose invasive fungal 541 infections are CT or MRI, since they provide overall information in less time [95] . However, there 542 is a need for multimodal imaging to overcome the limitations that exist for the individual use of 543 each imaging method (Table 1 ) [96] . The development of laboratory biological markers and the launching of antigen testing have 549 improved the diagnosis of invasive fungal infections regarding quickness and efficiency. Fungal 550 antigens, metabolites, or antibodies produced by the host's immune system can be detected in 551 several serum samples, but also urine and bronchoalveolar fluid [53] . In this review, the most 552 frequently used techniques will be presented. The developed assays use serum samples and rely on the activation of the LAL clotting cascade 561 that ultimately cleaves a chromogenic substance, p-nitroanilide, from a synthetic peptide in the 562 beta-glucan LAL, changing to a yellow colour. However, it has been observed that serum samples 563 have an inherent yellow colour that may overestimate quantification therefore, a variation of the β-564 (1,3)-D-glucan assay uses the diazo derivative of p-nitroanilide which by cleavage is purple [97] . (Serion GmbH, Germany) [110, 111] . In several studies with different designs and populations, the 606 clinical value of mannan detection assays showed variable sensitivity (52% to 85%) and specificity 607 (86.8% to 98%) [111] [112] [113] . The anti-mannan antibodies detection specificity and sensitivity ranged 608 from 57.7 to 80.4% and the specificity from 60 to 87% [112, 113] . These serologic tests present some disadvantages (Table 1) using CAGTA IFA IgG assay, and results showed a pooled sensitivity of 66%, and specificity of 625 76%, concluding that this assay has moderate accuracy for invasive candidiasis diagnosis [117] . CAGTA assay despite being easy handling, with a fast performance at low cost, this assay 627 is one of the few antibody-based that falls short in terms of sensitivity and specificity for invasive 628 candidiasis diagnosis, which ends up being a huge drawback (Table 1) . ELISA Platelia Aspergillus assay™ showed sensitivity values ranging from 44% to 100%, and 637 specificity from 78.6% to 100% [122, 123] . This assay has a higher sensitivity to Aspergillus non-638 fumigatus species, which turns out to be a drawback, because A. fumigatus is the prevalent pathogen 639 in invasive aspergillosis [35] . Despite GM being present in the cell wall of Histoplasma spp. and 640 Fusarium spp., this antigen detection assay is mentioned as an Aspergillus-specific methodology 641 [124] [125] [126] . However, this kit can be a useful tool for the diagnosis of infections caused by Fusarium 642 spp., since there is no antigen test for this pathogenic species [125] , and also for histoplasmosis, since Histoplasma spp. can take up to 4 weeks to grow in culture [50, 124, 126] . GM assay can be 644 a useful biomarker for the diagnosis of invasive aspergillosis. However, although being the most 645 frequently used assay in hospital settings for invasive aspergillosis, this assay has low sensitivity 646 for early diagnosis and falls short regarding specificity (Table 1) . (Table 1) . Several studies showed that rapid identification of the infectious agent leads to an 706 appropriate therapeutic plan, which results in lower mortality rates [31, 82, 135] . Since 1990, 707 thousands of studies referring to the diagnosis of fungal infections through molecular 708 methodologies have been published. However, the use of these techniques in hospital settings, for 709 some years was hampered by the lack of standardization and accreditation [136] . Molecular 710 methodologies have also evolved to be totally independent of the growth of the microorganism in 711 blood culture. The majority of molecular methodologies used in clinical context were first developed in 713 research laboratories and entitled "research use only" (RUOs) [136] . In order to reach bioindustry 714 and clinical laboratories, those methodologies must undergo a rigorous process of verification and 715 J o u r n a l P r e -p r o o f validation controlled by several entities [137] [138] [139] . Throughout the verification process, the new 716 method is defined, characterized, and compared with the gold standard methodology, considering 717 the disease or condition it aims to diagnose. This process allows the research centre to evaluate the 718 limitations, risks of error, and the likelihood of causing changes in the interpretation of the test 719 results or treatment decisions. The validation process incorporates the methodology quality control, 720 that is assessed during the time it is commercially available, to guarantee that it works the way it 721 was intended [137] [138] [139] . There is a special concern regarding the validation and verification of In clinical terms, PCR-based methodologies are commonly associated with the direct use 729 of samples from sterile sites such as whole blood and cerebrospinal fluid, or from nonsterile sites 730 like bronchoalveolar lavage, to detect fungal DNA (Fig. 2) . Nucleic acid amplification-based methodologies consist of enzymatic processes in which 732 one or more enzymes can synthesize copies of target sequences. That is achieved through a pair of 733 primers, which specifically bind to the target sequence, resulting in the amplification of that 734 sequence. The biggest drawback of these methods is contamination, which may lead to the 735 amplification of unwanted sequences [136] . PCR was the first nucleic acid amplification 736 methodology to be developed. Throughout the years, novel and more sophisticated variants of 737 conventional PCR have been developed, namely nested PCR, reverse transcriptase-PCR and real-738 time PCR. Regarding fungal pathogens detection, conventional PCR and real-time PCR are the most widely used, presenting high sensitivity, easy handling, and allowing identification of the 740 pathogen in a short time [35, 136, 140] . 741 For a few years, the scientific community faced some obstacles related to the manipulation 742 of PCR methodologies in hospital microbiology laboratories. For instance, the fungal burden 743 associated with invasive fungal infections was considered very close to the limit of detection of 744 PCR methodologies, so DNA extraction was a crucial step in the diagnosis [35, 136] . Fungi, In clinical contexts, the use of conventional PCR to detect and identify pathogenic 751 microorganisms is linked to an extra step for PCR product analysis, which increases the risk of 752 contamination by external factors. Another disadvantage is the lack of quantification of the PCR 753 products, precluding the differentiation between commensal colonization and active infection [35] . samples, no addition of reagents, or electrophoresis [35, 136, 143] . Several fluorescent reporters are 767 used to monitor qPCR reactions, being divided into intercalation dyes and probe-based qPCR 768 [136, 144] . However, intercalation dyes, like SYBR Green and EvaGreen, bind to any dsDNA, which is also 772 the case of primer-dimers. Nevertheless, these dyes are low-cost and prevent the need to resort to 773 probe design [136, [144] [145] [146] . The major downside associated with intercalation dyes qPCR, is that 774 an extra step is needed to perform amplicon analysis that is a melting curve analysis, which takes [136, 141, 147] . This approach is not the most indicated to medical diagnostics, and 782 so currently for a rigorous monitoring of the amplification in real-time, the probe-based qPCR is 783 used instead. Probe-based qPCR techniques are highly specific since they combine the specificities of 785 the primers and probes, and due to different dyes available, they can also be used in a multiplex the fluorescence is no longer quenched and can be measured [136, 145, 148] . Probe-based qPCR techniques can be applied in multiplex situations, although they depend 802 on the capacity of the equipment to read fluorescence at different wavelengths [136] . In this case, 803 each probe would be associated with the detection of a specific target, with a specific fluorescence, 804 and the equipment would have to be able to detect different fluorescence simultaneously [136, 147] . [136, 149] . In a study conducted by Camp and co-workers [149] , 813 the overall accuracy of Fungi Assay (real-time panfungal PCR) was compared with fungal cultures. For this study, 265 clinical samples (blood, CSF, BAL, and tissue) were used, and a 55.1% Europe have to comply with IVDR requirements, to achieve the "Conformité Européenne" (CE) 837 certification [150] . EU IVDR aims to regulate CE-IVDs (will have to be certified by notified bodies 838 and present post-market performance data) and comprises the (re)classification of both existing 839 and new IVDs using a risk-based system, which ranges from Class A (lowest risk) to Class D 840 (highest risk). Additionally, the laboratory's ability to manufacture and implement in-house IVDs 841 (IH-IVDs) will be more restricted under the EU IVDR, which will only allow the use of IH-IVDs are distinguished from specific signals referring to target cells. Finally, the sample is analysed using 894 fluorescence microscopy, in order to validate and examine the target cells [153, 154] . In a study conducted by Lies et al. [155] , solid-phase cytometry methodology was used to conditions therapies, and also in diagnostic approaches [161] . Sojinrin and co-workers [162] 952 developed a protocol to detect the presence of spore-forming fungi based on gold nanoparticles. Essentially, when the gold nanoparticles enter in contact, for example, with Aspergillus niger, they 954 endure structural and morphological changes, from spherical to star-shaped, and change of colour 955 J o u r n a l P r e -p r o o f from red to blue. This technique showed sensitivity of 80% and specificity of 95% for athlete's 956 foot diagnosis. This is a fast, straightforward and low-priced method, yet does not allow specific 957 identification of pathogens [162] . 960 Since 2001, NMR has been useful in the microbiology field for species identification and 961 detection, through the use of nanoparticles, with subsequent analysis by magnetic resonance [82] . In this case, the detection of the target organism is done by beads that have a complementary 963 sequence to the organism's DNA, allowing the binding. This binding allows the aggregation of 964 beads, which can be observed through magnetic resonance. NMR methodologies can be used alone, 965 or following a conventional PCR, for product analysis [34, 35] . T2Candida® was the first [82, 143] . Firstly, the clinical sample 970 is inserted into the platform, yielding an automated DNA extraction, which is then analysed by 971 magnetic resonance, detecting pathogenic Candida spp. [163] [164] [165] . In the clinical trial study, T2Candida® demonstrated a sensitivity of 91.1% and specificity of 99.4% which was a major 973 achievement regarding molecular diagnosis [165] . are designed as portable devices that convert biological and biochemical information into an output 978 analytical signal [166] . Fungal biosensors produced for clinic diagnosis have to fulfil several requirements, such as the careful selection of a specific biomarker of the target pathogen, which 980 has to be suitable for the biological recognition system and to hold measurable features associated 981 with normal conditions or with infection [166] . Pla et al. [167] described an innovative nanosensor 982 to detect C. auris based on biocompatible nanoporous anodic alumina (NAA) supports, with the 983 pores loaded with fluorophores and oligonucleotides attached. The oligonucleotides are specially 984 selected in order to make the sensor completely specific for C. auris. When this pathogen is present 985 in a sample, the oligonucleotide hybridizes to its genomic DNA exclusively, thus opening the pore 986 and releasing the trapped fluorophore. This system presented high sensitivity (85%) and selectivity 987 (100%) for C. auris detection from blood culture samples, also the results can be obtained within 988 an hour, and previous steps such as DNA extraction are not required [167] . Volatile organic compounds assay is a new type of methodology for the diagnosis of 992 invasive aspergillosis, with sensitivity rates above 90%. 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different methodologies used for identification of oral isolates of Candida species Comparison of 21-Plex PCR and API 20C AUX, MALDI-TOF MS, and rDNA sequencing for a wide range of clinically isolated yeast species: Improved identification by combining 21-Plex PCR and API 20C AUX as an alternative strategy for developing countries Evaluation of Common Commercial Systems for the Identification of High rates of misidentification of uncommon Candida species causing bloodstream infections using conventional phenotypic methods Rapid and reliable MALDI-TOF mass spectrometry identification of Candida non-albicans isolates from bloodstream infections Comparison of the accuracy of two conventional phenotypic methods and two MALDI-TOF MS systems with that of DNA sequencing analysis for correctly identifying clinically encountered yeasts Identification of Candida auris by use of the updated vitek 2 yeast identification system, version 8.01: A multilaboratory evaluation study Candida auris in Singapore: Genomic epidemiology, antifungal drug resistance, and identification using the updated 8.01 VITEK2 system Candida auris-associated Candidemia New clonal strain of Candida auris Identification of fungal isolates by MALDI-TOF mass spectrometry in veterinary practice: validation of a web application Development of a clinically comprehensive database and a simple procedure for identification of molds from solid media by matrix-assisted laser desorption ionization-Time of flight mass spectrometry Comparative evaluation of the Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems for non-albicans Candida and uncommon yeast isolates Evaluation of Autof MS 1000 and Vitek MS MALDI-TOF MS System in Identification of Closely-Related Yeasts Causing Invasive Fungal Diseases Evaluation of ID fungi plates medium for identification of molds by MALDI biotyper MALDI-TOF MS in a medical mycology laboratory: On stage and backstage Proof of Concept for MBT ASTRA, a Rapid Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS)-Based Method To Detect Caspofungin Resistance in Candida albicans and Candida glabrata Candida auris identification and rapid antifungal susceptibility testing against echinocandins by MALDI-TOF MS Diagnosis of bloodstream infections from positive blood cultures and directly from blood samples: recent developments in molecular approaches Seven years of clinical experience with the Yeast Traffic Light PNA FISH: Assay performance and possible implications on antifungal therapy Evaluation of the FilmArray Blood Culture Identification Panel: Results of a Multicenter Controlled Trial Clinical evaluation of the Filmarray blood culture identification panel in identification of bacteria and yeasts from positive blood culture bottles Multi-Center Evaluation of the BioFire® FilmArray® Blood Culture Identification 2 Panel for the Detection of Microorganisms and Resistance Markers in Positive Blood Cultures Evaluation of the Sepsis Flow Chip assay for the diagnosis of blood infections Clinical performance of the novel Genmark DX eplex blood culture ID Gram-positive panel Evaluation of Microbiological Performance and the Potential Clinical Impact of the ePlex® Blood Culture Identification Panels for the Rapid Diagnosis of Bacteremia and Fungemia Multicenter evaluation of a PCR-based digital microfluidics and electrochemical detection system for the rapid identification of 15 fungal pathogens directly from positive blood cultures Imaging spectrum of invasive fungal and fungal-like infections Visited on-line: october 10. Inside View Guidance on Imaging for Invasive Pulmonary Aspergillosis and Mucormycosis: From the Imaging Working Group for the Revision and Update of the Consensus Definitions of Fungal Disease from the EORTC/MSGERC Let's shine a light on fungal infections: A noninvasive imaging toolbox -3)-β-D-glucan assay: A review of its laboratory and clinical application Preemptive treatment of fungal infection based on plasma (1→3)β-D-glucan levels after liver transplantation Comparative performance evaluation of Wako β-glucan test and Fungitell assay for the diagnosis of invasive fungal diseases Multicenter clinical evaluation of the (1→3) β-D-glucan assay as an aid to diagnosis of fungal infections in humans Prospective survey of (1→3)-β-D-Glucan and Its Relationship to Invasive Candidiasis in the Surgical Intensive Care Unit Setting Contribution of Candida biomarkers and DNA detection for the diagnosis of invasive candidiasis in ICU patients with severe abdominal conditions Use and limits of (1-3)-β-D-glucan assay (fungitell), compared to galactomannan determination (platelia Aspergillus), for diagnosis of invasive aspergillosis Evaluation of a (1→3)-β-Dglucan assay for diagnosis of invasive fungal infections Evaluation of Wako beta-glucan test performance in diagnosing invasive fungal infections Comparative Analysis of the Wako -Glucan Test and the Fungitell Assay for Diagnosis of Candidemia and Pneumocystis jirovecii Pneumonia Difficulties in using 1,3-β-D-glucan as the screening test for the early diagnosis of invasive fungal infections in patients with haematological malignancies -High frequency of falsepositive results and their analysis Pseudomonas aeruginosa as a Cause of (1→3)-β-D-glucan Assay Reactivity How to interpret serum levels of betaglucan for the diagnosis of invasive fungal infections in adult high-risk hematology patients: optimal cut-off levels and confounding factors A Retrospective Assessment of Four Antigen Assays for the Detection of Invasive Candidiasis Among High-Risk Hospitalized Patients Clinical Practice Guideline for the Management of Candidiasis: 2016 Update by the Infectious Diseases Society of America Analysis of Clinical Characteristics and Diagnostic Value of Fungal Serology in Patients with Invasive Candidiasis Diagnostic value of Candida mannan antigen and anti-mannan IgG and IgM antibodies for Candida infection Visited on-line: october 28. Vircell Microbiologist Candida albicans Germ-Tube Antibody: Evaluation of a New Automatic Assay for Diagnosing Invasive Candidiasis in ICU Patients Performance of Candida albicans germ tube antibodies (CAGTA) and its association with (1 → 3)-β-D-glucan (BDG) for diagnosis of invasive candidiasis (IC) Diagnostic accuracy of Candida albicans germ tube antibody for invasive candidiasis: systematic review and meta-analysis Surveillance of Aspergillus galactomannan antigenemia for invasive aspergillosis by enzyme-linked immunosorbent assay in neutropenic patients treated for hematological malignancies Autopsycontrolled prospective evaluation of serial screening for circulating galactomannan by a sandwich enzyme-linked immunosorbent assay for hematological patients at risk for invasive aspergillosis Screening for circulating galactomannan as a noninvasive diagnostic tool for invasive aspergillosis in prolonged neutropenic patients and stem cell transplantation recipients: A prospective validation Comparison of serum galactomannan antigen detection and competitive polymerase chain reaction for diagnosing invasive aspergiliosis Evaluation of a Novel Aspergillus Antigen Enzyme-Linked Immunosorbent Assay Cross-reactivity of Aspergillus galactomannan in an HIV-infected patient with histoplasmosis Crossreactivity of Fusarium spp. in the Aspergillus galactomannan enzyme-linked immunosorbent assay False-positive Aspergillus galactomannan assay in solid organ transplant recipients with histoplasmosis Evaluation of real-time PCR, galactomannan enzyme-linked immunosorbent assay (ELISA), and a novel lateral-flow device for diagnosis of invasive aspergillosis Respiratory specimens and the diagnostic accuracy of Aspergillus lateral flow assays (LFA-IMMYTM): real-life data from a multicentre study Evaluation of the Performance of the IMMY sona Aspergillus galactomannan Lateral Flow Assay When Testing Serum To Aid in Diagnosis of Invasive Aspergillosis Diagnosis of Histoplasmosis Using the MVista Histoplasma Galactomannan Antigen Qualitative Lateral Flow-Based Immunoassay: A Multicenter Study Visited on-line: october 7. MiraVista Diagnostics Low cryptococcus antigen titers as determined by lateral flow assay should be interpreted cautiously in patients without prior diagnosis of cryptococcal infection Neglecting genetic diversity hinders timely diagnosis of cryptococcus infections Estimation of Direct Healthcare Costs of Fungal Diseases in the United States Molecular Detection and Identification of Fungal Pathognes Molecular Microbial Diagnostic Methods Molecular Diagnostic Assay Validation Update to the 2009 AMP Molecular Diagnostic Assay Validation White Paper Validation and Verification of Multiplex Nucleic Acid Assays Review: a comprehensive summary of a decade development of the recombinase polymerase amplification Detection of fungal pathogens by a new broad range real-time PCR assay targeting the fungal ITS2 region A comparison between CHROMagar, PCR-RFLP and PCR-FSP for identification of Candida species Nucleic Acid Tools for Invasive Fungal Disease Diagnosis Detection of invasive fungal pathogens by real-time PCR and high-resolution melting analysis Comparison of multiple DNA dyes for real-time PCR: Effects of dye concentration and sequence composition on DNA amplification and melting temperature Real-time PCR handbook Rapid identification of clinical common invasive fungi via a multi-channel real-time fluorescent polymerase chain reaction melting curve analysis Mode of action and application of Scorpion primers to mutatation detection Clinical evaluation of an in-house panfungal real-time PCR assay for the detection of fungal pathogens The New EU Regulation on In Vitro Diagnostic Multiplex PCR based strategy for detection of fungal pathogen dna in patients with suspected invasive fungal infections Detection and quantification of bacteria and fungi using solid-phase cytometry Rapid and direct quantification of viable Candida species in whole blood by use of immunomagnetic separation and solid-phase cytometry Rapid detection and quantification of Aspergillus fumigatus in environmental air samples using solid-phase cytometry How to Read and Interpret FTIR Spectroscope of Organic Material FTIR and raman spectroscopy-based biochemical profiling reflects genomic diversity of clinical candida isolates that may be useful for diagnosis and targeted therapy of candidiasis Yelena Souprun MH. FTIR microscopy as a method for identification of bacterial and fungal infections Enhancing disease diagnosis: Biomedical applications of surface-enhanced raman scattering Rapid detection method for pathogenic Candida captured by magnetic nanoparticles and identified using SERS via AgNPs+ Surface chemistry of gold nanoparticles for health-related applications Plasmonic gold nanoparticles for detection of fungi and human cutaneous fungal infections T2 magnetic resonance for fungal diagnosis T2 magnetic resonance enables nanoparticle-mediated rapid detection of candidemia in whole blood T2 magnetic resonance assay for the rapid diagnosis of candidemia in whole blood: A clinical trial Biosensors and Diagnostics for Fungal Detection Oligonucleotide-capped nanoporous anodic alumina biosensor as diagnostic tool for rapid and accurate detection of Candida auris in clinical samples Electronic nose technology for detection of invasive pulmonary aspergillosis in prolonged chemotherapy-induced neutropenia: A proof-of-principle study Comparisons between mammalian and artificial olfaction based on arrays of carbon black-polymer composite vapor detectors Profiling of volatile organic compounds produced by clinical Aspergillus isolates using gas chromatography-mass spectrometry Detection of 2-Pentylfuran in the breath of patients with Aspergillus fumigatus Detection of characteristic metabolites of Aspergillus fumigatus and Candida species using ion mobility spectrometry -metabolic profiling by volatile organic compounds Lion T. Human Fungal Pathogen Identification: Methods and Protocols Alastruey-izquierdo A. Diagnosis of Breakthrough Fungal Infections in the Clinical Mycology Laboratory : An ECMM Consensus Statement Recent advances in the microbiological diagnosis of bloodstream infections Mass Spectometry for Microorganism Identification Serology anno 2021-fungal infections: from invasive to chronic The use of mannan antigen and anti-mannan antibodies in the diagnosis of invasive candidiasis Molecular diagnostics in medical mycology Performance of the lightCycler septiFast test M grade in detecting microbial pathogens in purulent fluids Detection of Aspergillus fumigatus in blood samples from critically ill patients in intensive care units by use of the septifast assay Diagnostic performance of a novel multiplex PCR assay for candidemia among ICU patients Evaluation of the MagicplexTM sepsis real-time test for the rapid diagnosis of bloodstream infections in adults Molecular methods for the diagnosis of invasive candidiasis Evaluation of Two Commercial Real-Time PCR Kits for Aspergillus DNA Detection in Bronchoalveolar Lavage Fluid in Patients with Invasive Pulmonary Aspergillosis Evaluation of MycAssayTM Aspergillus for Diagnosis of Invasive Pulmonary Aspergillosis in Patients without Hematological Cancer Overview of commercially available PCR assays for the detection of Aspergillus spp Validation of a new Aspergillus real-time PCR assay for direct detection of Aspergillus and azole resistance of Aspergillus fumigatus on bronchoalveolar lavage fluid PCR-based detection of Aspergillus fumigatus Cyp51A mutations on bronchoalveolar lavage: A multicentre validation of the AsperGenius assay® in 201 patients with haematological disease suspected for invasive aspergillosis Analytical and clinical evaluation of the pathonostics aspergenius assay for detection of invasive aspergillosis and resistance to azole antifungal drugs directly from plasma samples Evaluation of three commercial PCR assays for the detection of azole-resistant Aspergillus fumigatus from respiratory samples of immunocompromised patients Evaluation of Real Time PCR Aspergillus spp. in bronchoalveolar lavage samples Visited on-line: march 30 A New Age in Molecular Diagnostics for Invasive Fungal Disease: Are We Ready? Molecular diagnosis of invasive aspergillosis and detection of azole resistance by a newly commercialized PCR kit Methods for Detection of Aspergillus fumigatus Resistance in Clinical Samples Evaluation of the new AspID polymerase chain reaction assay for detection of Aspergillus species: A pilot study Validation and implementation of a commercial real-time PCR assay for direct detection of Candida auris from surveillance samples Comparative evaluation between the RealStar Pneumocystis jirovecii PCR kit and the AmpliSens Pneumocystis jirovecii (carinii)-FRT PCR Kit for detecting P. Jirovecii in Non-HIV immunocompromised patients Evaluation of a new commercial real-time PCR assay for diagnosis of Pneumocystis jirovecii pneumonia and identification of dihydropteroate synthase (DHPS) mutations Serial detection of circulating mucorales DNA in invasive mucormycosis: A retrospective multicenter evaluation Evaluation of MucorGenius® mucorales PCR assay for the diagnosis of pulmonary mucormycosis